GSK3 polypeptides

ABSTRACT

The invention provides truncated GSK3 polypeptides capable of crystallization, including GSK 3 α and GSK 3 β polypeptides, and use of these polypeptides to identify and optimize GSK3 inhibitors. Also provided are GSK3 polypeptides having at least one substituted amino acid that differs from wild-type GSK3, wherein the substituted amino acid is incapable of being phosphorylated. The invention finds use in providing methods of identifying and optimizing compounds useful for treating diseases mediated by GSK3 activity, including Alzheimer&#39;s disease, type 2 diabetes, and inflammation.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional PatentApplication No. 60/221,242 filed Jul. 27, 2000, where this provisionalapplication is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention provides materials and methods relating toidentification and optimization of selective inhibitors of glycogensynthase kinase 3 (GSK3), and also relates to methods of treating acondition mediated by GSK3 activity. Such conditions include Alzheimer'sdisease, type 2 diabetes, and inflammation.

[0004] 2. Description of the Related Art

[0005] Glycogen synthase kinase 3 (GSK3) is a proline-directedserine/threonine kinase originally identified as an activity thatphosphorylates glycogen synthase as described in Woodgett, TrendsBiochem Sci. 16:177-181 (1991). The role in glucose metabolism has beenelaborated recently in Summers et al., J. Biol. Chem. 274:17934-17940(1999). GSK3 consists of two isoforms, α and β, and is constitutivelyactive in resting cells, inhibiting glycogen synthase by directphosphorylation. Upon insulin activation, GSK3 is inactivated, therebyallowing the activation of glycogen synthase and possibly otherinsulin-dependent events. GSK3 is inactivated by other growth factors orhormones that, like insulin, signal through receptor tyrosine kinases.Examples of such signaling molecules include IGF-1 and EGF as describedin Saito et al., Biochem. J. 303:27-31 (1994), Welsh et al., Biochem. J.294:625-629 (1993), and Cross et al., Biochem. J. 303:21-26 (1994). GSK3has been shown to phosphorylate β-catenin as described in Peifer et al.,Develop. Biol 166:543-56 (1994). Other activities of GSK3 in abiological context include GSK3's ability to phosphorylate tau proteinin vitro as described in Mandelkow and Mandelkow, Trends in Biochem.Sci. 18:480-83 (1993), Mulot et al., Febs Lett 349: 359-64 (1994), andLovestone et al., Curr. Biol. 4:1077-86 (1995), and in tissue culturecells as described in Latimer et al., Febs Lett 365:42-6 (1995).Selective inhibition of GSK3/may be useful to treat or inhibit disordersmediated by GSK3 activity.

[0006] There is a need in the art for compositions and molecules thatbind to or interact with GSK3, thereby mediating GSK3 activity. Theinvention meets this need by providing crystallizable GSK3 polypeptidesuseful for design and optimization of GSK3 inhibitors.

BRIEF SUMMARY OF THE INVENTION

[0007] The invention provides GSK3β molecules with N- and C-terminaltruncations, wherein the molecules are capable of crystallization.

[0008] The invention further provides GSK3β molecules truncated at aminoacid R³⁴⁴, R³⁵⁴, T³⁶⁴, A³⁷⁴, and I³⁸⁴.

[0009] The invention provides a polypeptide consisting essentially ofSEQ ID NO:2 or SEQ ID NO:3, polynucleotides encoding these polypeptides,and vectors comprising these polynucleotides.

[0010] The invention still further provides GSK3β molecules whereintranslation of the molecule begins at G³⁴, T³⁹, P⁴⁴, D⁴⁹ or V⁵⁴.

[0011] The invention also provides GSK3α molecules with N- andC-terminal truncations, wherein the molecules are capable ofcrystallization.

[0012] The invention further provides a GSK3α molecule whereintranslation of the molecule begins at S⁹⁷ and ends at S⁴⁴⁷,polynucleotides encoding this polypeptide, and vectors comprising thesepolynucleotides.

[0013] The invention further provides a method of identifying a GSK3polypeptide capable of crystallization, comprising: (a) providing atruncated GSK3 polypeptide; (b) testing the polypeptide for formation ofcrystals.

[0014] The invention also provides GSK3 polypeptides capable ofinteracting with inhibitors of GSK3.

[0015] The invention further provides a method of identifying anenzymatically active GSK3 polypeptide, comprising: (a) providing atruncated GSK3 polypeptide; (b) contacting the polypeptide with asubstrate of GSK3; and (c) measuring the kinase activity of thepolypeptide after contacting the polypeptide with the substrate, whereinthe polypeptide is active if it shows >0.01× the activity of thefull-length enzyme and preferably >0.1× the activity of the full-lengthenzyme.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE IDENTIFIERS

[0016]FIG. 1 provides the polypeptide sequence of human GSK3β (SEQ IDNO:1).

[0017]FIG. 2 provides the polypeptide sequence of truncated GSK3βpolypeptide 557 (SEQ ID NO:2). The first ten amino acids represent aGlu-tag, followed by a Gly linker before Met at position 1.

[0018]FIG. 3 provides the polypeptide sequence of truncated GSK3βpolypeptide 580 (SEQ ID NO:3). The first ten amino acids represent aGlu-tag, followed by a Gly linker before Gly at position 34.

[0019]FIG. 4 provides the polypeptide sequence of human GSK3α (SEQ IDNO:4).

[0020]FIG. 5 provides the polypeptide sequence of human GSK3α truncatedat position 447 (SEQ ID NO:5).

[0021]FIG. 6 provides the polypeptide sequence of human GSK3α truncatedat position 97 (SEQ ID NO:6).

[0022]FIG. 7 provides the polypeptide sequence of human GSK3α fromposition 97 to position 447 (SEQ ID NO:7).

DETAILED DESCRIPTION OF THE INVENTION

[0023] The invention provides materials and methods for identifying andoptimizing inhibitors of GSK3, including GSK3α and GSK3β. The providedmaterials include C- and N-terminal truncated GSK3β molecules that arecapable of crystallization and may, but need not, retain GSK3 kinaseactivity, preferably more than 0.01× the activity of the full-lengthenzyme and more preferably more than 0.1× the activity of thefull-length enzyme. There is a need in the art for such inhibitors, inview of the role of GSK3 in a variety of diseases and conditions,including Alzheimer's disease, type 2 diabetes and inflammation. Suchinhibitors can be identified, and identified inhibitors can beoptimized, using the crystallizable GSK3 polypeptides of the invention.

[0024] The invention provides a variety of GSK3β polypeptides thatdiffer from the native polypeptide at the C- and/or N-terminus. Theamino acid sequence of GSK3β is shown in FIG. 1 (SEQ ID NO:1). Includedwithin the scope of the invention are any and all truncations of GSK3βpolypeptide wherein the truncated polypeptide is capable ofcrystallization and may, but need not, retain kinase activity asmeasured using the kinase assays described herein. Persons of skill inthe art will realize that limited mutation of the protein, or certainpost-translational modifications, might be sufficient to inactivate thekinase yet retain the essential 3D structure. Such inactive butstructurally related molecules would also be useful for the design andoptimization of inhibitors. Kinase assays are disclosed in U.S. Pat.Nos. 6,057,117 and 6,057,286, which are incorporated herein byreference. The percent activity that is retained, if any, is notcrucial. Methods of assaying activity in the presence and absence of aninhibitor are described herein.

[0025] The invention provides numerous truncated GSK3β polypeptides thatmeet these criteria. A preferred polypeptide is designated BV557 inwhich the C-terminal amino acid is R³⁸⁴. This molecule has beensuccessfully crystallized. Additional active polypeptides include thosewith truncations at amino acid R³⁴⁴, R³⁵⁴, A³⁷⁴, and I³⁸⁴.

[0026] The invention also provides truncated GSK3α polypeptides,including a GSK3α polypeptide beginning at S⁹⁷ and ending at S⁴⁴⁷.

[0027] Additional truncated GSK3 polypeptide include those beginningwith an N-terminal amino acid that differs from that of the nativeprotein in that 1 or more amino acids are deleted from the N-terminus.Preferred N-terminal truncations include GSK3β molecules whereintranslation of the molecule begins at G³⁴, T³⁹, P⁴⁴, D⁴⁹ or V⁵⁴. Anexample is BV580 (amino acids 34 to 384) which has been crystallized.

[0028] The invention is not limited to these disclosed truncatedmolecules. Using the methods and assays described herein, one of skillcan construct additional truncated molecules, such as those having 36-76amino acids deleted from the C-terminus, and/or 35-54 amino acidsdeleted from the N-terminus. Such deletions can occur individually, or apolypeptide can have both an N-terminal deletion and a C-terminaldeletion. It is preferable but not necessary that the kinase domainremain relatively intact as reflected by the detection of enzymaticactivity, such as by using the assays described herein. It is alsodesirable, although not essential, that the enzymatic activity becapable of inhibition by a known GSK3 inhibitor, such as lithium. Atruncated molecule meeting these criteria will be suitable for testingGSK3 inhibitors as potential therapeutic agents, and for optimizing GSK3inhibitors.

[0029] A truncated GSK3β polypeptide of the invention can consist ofbetween about 250 and 419 contiguous amino acids of SEQ ID NO:1;preferably between about 278 and 419 contiguous amino acids of SEQ IDNO:1; more preferably between about 285 and 384 contiguous amino acidsof SEQ ID NO:1; and most preferably between about 351 and 384 contiguousamino acids of SEQ ID NO:1. Preferred truncated GSK3β polypeptidesinclude those beginning at amino acid 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62 of SEQ IDNO:1, and ending at amino acid 340, 341, 342, 343, 344, 345, 346, 347,348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361,362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375,376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389,390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403,404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417,418 or 419 of SEQ ID NO:1. The polypeptide can begin with any one of thelisted beginning amino acids and end with any one of the ending aminoacids. Exemplary and non-limiting embodiments begin at amino acid 34,39, 44 or 54 and end at amino acid 420. Other particularly preferredembodiments begin at about amino acid 1 and end at amino acid 340, 344,354, 374, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409,410, 411, 412, 413, 414, 415, 416, 417, 418, 419, or 420.

[0030] The truncated GSK3α polypeptide of the invention can consist ofbetween about 182 and 482 contiguous amino acids of SEQ ID NO:4,preferably between about 182 and 386 contiguous amino acids of SEQ IDNO:4, more preferably between about 182 and 351 contiguous amino acidsof SEQ ID NO:4, and most preferably from about S⁹⁷ to S⁴⁴⁷ of SEQ IDNO:4.

[0031] The truncated GSK3 polypeptides can be prepared by any methodknown in the art. One method involves expression of a suitably preparedpolynucleotide encoding a polypeptide having the desired truncation. Forexample, a preferred polypeptide of the invention, BV557, was preparedby creating a construct encoding GSK3β starting at M¹ and ending atI³⁸⁴, as described in the Examples. Briefly, insect cells weretransfected with baculovirus vector (designatedpBlueBac4.5.Glu.GSK3B.DC.I384#28), which encodes BU557, and the proteinwas extracted from the lysed cells. The protein was purified by affinitychromatography using an anti glu-tag monoclonal antibody immobilized ona Sepharose column. Activity of the purified protein was assayed usingthe in vitro kinase assay described in U.S. Pat. No. 6,057,286.

[0032] The Examples herein describe the production of BV557, BV580, andother truncated GSK3 polypeptides by expression of vectors encoding thepolypeptides, followed by isolation and purification of thepolypeptides. The polypeptide can also be produced by enzymatic cleavageof a native GSK3 protein, using methods known in the art. Other suitablemethods include expression of a polynucleotide encoding a truncatedpolypeptide in a variety of cell types, including mammalian, bacterial,or yeast cells. However, the preferred cell for expression of thepolypeptide is an insect cell, preferably a baculovirus-infectableinsect cell, such as a Sf9 cell.

[0033] The invention also provides unphosphorylated forms of GSK3wherein the ATP binding site is identical to that of the wild-typeprotein. Such forms include Y216 non-phosphorylated GSK3β and Y279non-phosphorylated GSK3α. Other forms include constructs with at leastone amino acid change that prevents phosphorylation, such as GSK3β inwhich Y216 is changed to F216, and GSK3α in which Y279 is changed toF279. These forms are suitable for inhibitor binding assays to identifyinhibitors of GSK3. The invention provides a GSK3β molecule in whichposition 216 is not phosphorylated. We have demonstrated that a GSK3βpeptide with Y216 mutated to F216 crystalized and exhibits a structurein which the ATP-binding site is not substantially different from theun-mutated peptide.

[0034] Additional single and multiple amino acid changes include S⁹ toA⁹ in GSK3β and S²¹ to A²¹ in GSK3α.

[0035] These changes in phosphorylation, or ability to bephosphorylated, are optionally incorporated into the truncated forms ofGSK3α a and GSK3β disclosed herein.

[0036] The invention therefore provides GSK3 molecules suitable fordesign and optimization of inhibitors of GSK3 as pharmaceutical agents.

[0037] The GSK3 constructs of the invention are capable ofcrystallization. In purified form the constructs bind to inhibitors in amanner that is comparable to inhibitor binding to the native GSK3polypeptide, due to the retention of the correct folding conformation atthe inhibitor binding site. Potential to crystallize is measured using avariety of assays including specific activity, aggregation,microheterogeneity. (See, for example, Table 1). These parameters areindicative of the purity of the preparation and of the solubility of theconstruct. The specific activity is also a preferred assay for detectingbinding of an inhibitor to the correct binding site of the GSK3construct. Another suitable method is fluorescence polarization.Briefly, a putative inhibitor, with an attached fluorophore, tumblesfreely in solution. Thus when the fluorophore is excited by polarizedlight, the emitted light which is produced after a finite delay now hasrandom polarity and the emitted light is no longer polarized. In thepresence of a GSK3 construct with an intact inhibitor binding site, thetumbling rate is slowed sufficiently to ensure that, even though thelight emission is delayed with respect to the excitation, thefluorophore has only moved very slightly. Thus, the excited lightmaintains polarization. A measurement of fluorescence polarizationtherefore indicates whether or not the GSK3 construct is suitable foridentifying and optimizing an inhibitor. The fluorophore can be attachedto a compound such as staurosporine (ICN Pharmaceuticals, Inc., CostaMesa, Calif.). GSK3 constructs may not retain kinase activity, but theirinhibitor binding can still be assessed using fluorescence polarizationassays.

[0038] The term “truncated glycogen synthase kinase 3” or “truncatedGSK3” as used herein refers to GSK3α or GSK3β. GSK3 is a proteinoriginally identified by its phosphorylation of glycogen synthase asdescribed in Woodgett et al, Trends Biochem Sci, 16:177-181 (1991).Synonyms of GSK3 are tau protein kinase I (TPK I), FA kinase and kinaseFA. Mammalian forms of GSK3 have been cloned as described in Woodgett,EMBO J. 9(8):2431-2438 (1990). Inhibitors of truncated GSK3 polypeptidescan be inhibitors of any of the known forms of GSK3, including eitherGSK3α or GSK3β or both. Truncated polypeptides of the invention possessone or more of the bioactivities of the GSK3 protein, including kinaseactivities such as polymerizing tau protein, or phosphorylating glycogensynthase, for example. Thus, truncated GSK3 polypeptides useful fordesigning and optimizing inhibitors of GSK3 can have sequence identityof at least 40%, preferably 50%, preferably 60%, preferably 70%, morepreferably 80%, and most preferably 90% to the amino acid sequence ofthe native protein, wherever derived, from human or nonhuman sources.The polynucleotides encoding a GSK3 polypeptide can have 60%, preferably70%, more preferably 80%, more preferably 90% and most preferably 95%sequence identity to a native polynucleotide sequence of GSK3. Alsoincluded, therefore, are alleles and variants of the nativepolynucleotide sequence such that the polynucleotide encodes an aminoacid sequence with substitutions, deletions, or insertions, as comparedto the native sequence.

[0039] The term “peptide substrate” refers to a peptide or a polypeptideor a synthetic peptide derivative that can be phosphorylated by GSK3activity in the presence of an appropriate amount of ATP or a phosphatedonor. Detection of the phosphorylated substrate is generallyaccomplished by the addition of a labeled phosphate that can be detectedby some means common in the art of labeling, such as radiolabeledphosphate. The peptide substrate may be a peptide that resides in amolecule as a part of a larger polypeptide, or may be an isolatedpeptide designed for phosphorylation by GSK3.

[0040] As disclosed in U.S. Pat. Nos. 6,057,117 and 6,057,286, in vitromethods of assaying GSK3 activity include constructing peptidesubstrates. The peptide substrate can be any peptide substratephosphorylatable by GSK3, and may be a peptide substrate including theformula: anchor ligand-(X)_(n)SXXXS(X)_(m) (SEQ ID NO:8) (wherein X isany amino acid, n is any integer, m is any integer, and preferablyn+m+5<20, i.e. n+m<15) prephosphorylated at the C terminal serine. Theassay is performed by contacting the prephosphorylated substrate withtruncated GSK3 polypeptide in the presence of radiolabeledγphosphate-ATP, a substrate anchor, and optionally a candidateinhibitor. The in vitro method of identifying an inhibitor of GSK3kinase activity includes contacting a peptide substrate coupled to ananchor ligand with truncated GSK3 polypeptide in the presence ofradiolabeled γphosphate-ATP, a substrate anchor, and candidateinhibitor, measuring an incorporation of radiolabel into the peptidesubstrate, then, in a separate assay vessel contacting a peptidesubstrate coupled to an anchor ligand with truncated GSK3 in thepresence of radiolabeled γphosphate-ATP, and a substrate anchor, andmeasuring incorporation of radiolabel into said peptide substrate;ultimately an inhibitor of truncated GSK3 kinase activity is identifiedby a reduction of label incorporation in the assay with the candidateinhibitor as compared to the assay without the candidate inhibitor.

[0041] To conduct the in vitro kinase assay of the invention usingmicrowells, scintillant may be present by precoating the wells with ascintillant material, or by adding it later following a wash step, asdescribed in Example 4. The scintillant can be obtained from Packard,Meridian, Conn. Wells coated with scintillant are then in additioncoated with streptavidin as a substrate anchor, where biotin is theanchor ligand on the peptide. Alternatively, the streptavidin can bepresent on agarose beads containing scintillant or may be coated on anotherwise untreated plate to which scintillant is added subsequently. Inany event, the streptavidin in the wells binds the biotin that contactsit. Following an assay using radiolabeled ATP, the radiolabelincorporated into the phosphorylated substrate that has been conjugatedto the biotin will cause the scintillant to emit light. Where thestreptavidin is attached to agarose beads containing scintillant,binding a biotin-conjugated radiolabeled peptide substrate will causethe beads to scintillate. In both the case of the wells lined with thescintillant, and the agarose beads containing scintillant, a reductionin scintillation as compared to a control amount of scintillationmeasured under non-inhibitory conditions, indicates the presence of afunctional inhibitor of GSK3 activity. If the peptide has beenphosphorylated by GSK3 with ³²P-labeled or ³³P-labeled phosphate,radioactive decay will cause the scintillant present in a microwell ormixed in agarose beads that are present in the reaction mixture to emitlight and the measure of the amount of light emitted will be a measureof the activity of GSK3 in the assay. Low activity of GSK3 observed inthe presence of a candidate inhibitor, as compared to the activity ofGSK3 in the absence of the inhibitor, may indicate that the inhibitor isfunctional and can inhibit GSK3 kinase activity. In any case, an excessof streptavidin over peptide should be loaded into each well or shouldbe affixed to the agarose beads.

[0042] GSK3 inhibitory activity can be measured using a cell-free assayas disclosed in publication WO 99/65897, and as described in Example 4herein. Activity can also be measured using a cell-based assay. Briefly,a cell line, such as a Cos cell line, is transfected with Tau and with aGSK3 polypeptide. The phosphorylation of Tau at a specific site ismonitored using a monoclonal antibody, as phosphorylation at that siteis dependent on GSK3 activity.

[0043] Exemplary polypeptides of the invention include the followingtruncated polypeptides with reference to SEQ ID NO:1:

[0044] GSK3β truncated at R³⁴⁴

[0045] GSK3β truncated at R³⁵⁴

[0046] GSK3β truncated at T³⁶⁴

[0047] GSK3β truncated at A³⁷⁴

[0048] GSK3β truncated at I³⁸⁴

[0049] GSK3β beginning at G³⁴

[0050] GSK3β beginning at T³⁹

[0051] GSK3β beginning at p⁴⁴

[0052] GSK3β beginning at D⁴⁹

[0053] GSK3β beginning at V⁵⁴

[0054] The above truncations can be combined, providing a GSK3βpolypeptide beginning at any of G³⁴, T³⁹, P⁴⁴, D⁴⁹, or V⁵⁴, and endingat any of R³⁴⁴, R³⁵⁴, T³⁶⁴, A³⁷⁴, or I³⁸⁴.

[0055] Other exemplary polypeptides of the invention include thefollowing truncated polypeptides with reference to SEQ ID NO:4:

[0056] GSK3α truncated at S⁴⁴⁷

[0057] GSK3α beginning at S^(97.)

[0058] GSK3α beginning at S⁹⁷ and truncated at S⁴⁴⁷.

[0059] A truncated GSK3 polypeptide of the invention can be selected onthe basis of one or more parameters. A polypeptide will preferablycrystallize in a form that is similar to that of native GSK3, withcorrect folding at and around the inhibitor binding site.Crystallization can be performed using a Crystal Screen Kit (HamptonResearch, Laguna Niguel, Calif.), or methods described by Jancarik, J.et al., J Appl. Cryst. 24:409-411, 1991. The potential of a polypeptideto form crystals can be evaluated on the basis of specific activity,purity, homogeneity, mass spectrometry, aggregation, and dynamic lightscattering. A preferred truncated polypeptide will meet the followingparameters: purity of at least 90%; less than 100% aggregation at 4° C.at two weeks; and less than 50% heterogeneity (50% or greater of thedesired form). A most preferred truncated polypeptide will have a purityof at least 98%, no aggregation at 4° C. at two weeks; and less than 5%heterogeneity (unphosphorylated form). Such parameters indicate that thepolypeptide preparation is likely to crystallize, making it suitable fordiscovering and optimizing GSK3 inhibitors.

[0060] A prerequisite for crystallization is to obtain a sufficientlyconcentrated stock of protein. Not all GSK3 constructs will remainsoluble at the required concentration. A preferred concentration is >1mg/ml, more preferred is >5 mg/ml, and most preferred is >10 mg/ml.

[0061] The polypeptides disclosed herein as 557 (SEQ ID NO:2), 580 (SEQID NO:3), 458, and 524 meet the criteria described above (see Example3). Polypeptide 458 consists of amino acids 1-420 of SEQ ID NO:1 plusthe following addition at the N-terminus: EFMPTEAMAAPKRVI (SEQ ID NO:8).Polypeptide 524 consists of amino acids 1-420 of SEQ ID NO:1 plus thefollowing addition at the N-terminus: EYMPMEGGG (SEQ ID NO:9). Othermodified or truncated GSK3 polypeptides can be prepared and tested asdescribed herein.

EXAMPLES

[0062] The following examples are exemplary only, and are not intendedto limit the invention.

Example 1 Preparation and Purification of GSK3β Construct 557

[0063] Lysis and Extraction. Insect cell slurry from Sf9 cells (about 10g) from a 1 liter flask growth was combined with 30 ml of lysate buffer:20 mM Tris, pH 8.0/80 mM NaCl/1 mM MgCl₂/1 mM Arsenate/1 mMTungstenate/1 mM PMSF/ 0.5 mg Leupeptin/0.2 mg Aprotinin. Cells werelysed using a Dounce homogenizer. Improved extraction of the protein wasaccomplished by the addition of 5% glycerol and 0.2% octylglucoside. Themixture was allowed to stir, on ice, for 30 minutes. The total lysatewas centrifuged at 39000×g for 25 minutes at 4° C. The resultingsupernatant contained the extracted GSK3-β#557.

[0064] Ion Exchange Chromatography. The following materials andconditions were used: The resin was Fractogel EMD SO₃-(M); the columndiameter was 1.6 cm and the column volume was 10 ml. The column was runat a flowrate of 90 cm/hour using equilibration buffer of 20 mM NaPhosphate/5% Glycerol, pH 7.5. Chromatography was carried out at 4° C.

[0065] The lysate supernatant was diluted 1:1 with S-fractogelequilibration buffer, and loaded onto the equilibrated column. Thecolumn was washed with a total of 14 column volumes of equilibrationbuffer. The GSK3-β was eluted with a linear salt gradient, over 20column volumes, to equilibration buffer plus 1M NaCl. 3 ml/fraction wascollected during gradient elution. The pool was made based on SDS-PAGEand Western blot results of the fractions collected. Fractions 13-24were pooled.

[0066] Affinity Chromatography was performed using the followingmaterials and procedures: The resin was anti glu-tag monoclonal antibodyimmobilized onto Protein G Sepharose, and the equilibration buffer wasPBS/0.3M NaCl/0.2% octylglucoside/10% Glycerol. The column diameter was1.6 cm and the column volume was 13 ml. The flow rate was 30 cm/hourduring load and wash, and 15 cm/hour during elution.

[0067] The S-Fractogel pool was loaded at 30 cm/hour onto equilibratedcolumn. The column was washed down to absorbance baseline withapproximately 6 column volumes of equilibration buffer, and GSK3β waseluted with 50 ml of equilibration buffer containing 2 mg of elutionpeptide (EYMPTD). The flow rate during elution was lowered to 15 cm/hour. 2 ml/fractions were collected during the elution. Based onSDS-PAGE results, elution fractions 6-17 were pooled with a total volumeof 24 ml.

[0068] Final Yield. The affinity column pool, at a concentration of 0.17mg/ml, contained 4.1 mg of GSK3β#557. This translates to a final yieldof 4.1 mg purified 557/liter of growth. Purity, after this 2 columnpurification, was estimated at >95% by visual inspection of SDS-PAGEresults.

Example 2 Preparation and Purification of GSK3β Construct 580

[0069] Extraction. SF9 cell paste from a 10 L fermentation was washedwith 100 mL PBS (10 mM NaPi, pH7.5, 150 mM NaCl) and then resuspendedwith 300 mL of Buffer H (20 mM Tris, pH 7.5, 1 mM Tungstate, 1 mMArsenate, 5 mM DTT, 10 μg/mL Leupeptin, 1 μg/mL pepstatin A, 10%glycerol, 0.35% Octyl glucoside, 1 mM Mg²⁺). Cells were homogenized in a100-mL Dounce Homogenizer (20 strokes with pestle B). The combinedhomogenate was centrifuged in a Ti45 rotor at 40,000 rpm for 35 minutesto remove cell debris and nuclei. The supernatant from thecentrifugation were carefully decanted and filtered through 0.45μfilter.

[0070] S-Fractogel. 100 mL S-Fractogel (EM Science, Cat#18882) waspacked into a 3.2 cm×12.5 cm column and equilibrated with>1 L of bufferA (20 mM Tris, pH 7.5, 10% glycerol). The filtrate from the previousstep was loaded at 15 mL/min onto the column. The column was washed with1 L of buffer A and then eluted with a linear gradient from 0 to 1 MNaCl in buffer A over 20 column volumes. The eluant was fractionatedinto 20 mL each. Fractions containing GSK3 were detected by Western Blotusing anti-GSK antibody (Santa Cruz Biotech, Cat #SC-7291). TheWestern-Blot positive fractions were pooled and mixed with equal volumeof buffer M (20 mM Tris, pH 7.5, 10% glycerol, 3.1 M NaCl) and filteredthrough a 0.45μ filter. The filtrate was saved for Phenyl-650 Mchromatography.

[0071] Phenyl-650 M. 37.5 mL Phenyl-650 M (Tosohass, Cat #014943) waspacked into a 2.2×10 cm column and equilibrated with 500 mL of buffer C(20 mM Tris, pH 7.5, 10% glycerol, 1.6 M NaCl). Filtrate fromS-fractogel step was loaded onto the column at 7.5 mL/min. After theloading was completed, the column was washed with 6.5 cv buffer C andeluted with a linear gradient from 0% to 100% Buffer D (20 mM Tris, pH7.5, 10% glycerol) over 20 column volumes. Fractions were collected at15 mL each and GSK containing fractions were detected by Western Blotusing anti-GSK antibody. The Western positive fractions were pooled andloaded onto a Glu-tag antibody affinity column.

[0072] Glu-tag antibody Affinity Chromatography. Use of a Glu-tag isdescribed in Rubinfeld et al., Cell 65:1033-1042, 1991, and a hybridomaexpressing anti-Glu-tag antibody is described in Grussenmyer et al.,PNAS 82:7952-7954 (1985). 50 mg of the Glu-tag antibody was immobilizedonto 25 mL of Affi-Gel 10 (BioRAD, Cat #153-6046) and packed into a2.2×6.5 cm column. The column was equilibrated with 200 mL of buffer E(20 mM Tris, pH 7.5, 10% glycerol, 0.3 M NaCl, 0.2% Octylglucoside) andthe fraction pool from the Phenyl-650 M step was loaded at 1.0 mL/min.After the loading was completed, the column was washed with 100 mL ofbuffer E and then eluted with 60 mL Glu-tag peptide (100 μg/mL) inBuffer E and fractionated into 5 mL each. GSK containing fractions weredetected with SDS-PAGE and Coomassie Blue staining. These fractions werepooled and concentrated to approximately 6 mg/mL in an Amiconconcentrator using a 10 k MWCO YM10 membrane. The concentrated materialwas then ready for crystallization.

Example 3 Activity of Trumcated GSK3β Polypeptides

[0073] A reaction mixture was prepared containing 5.9 μMprephosphorylated SGSG-linked CREB peptide (Wang et al., Anal Biochem.,220:397-402 (1994))μ in reaction buffer (5 mM Tris, pH 7.5, 5 mM DTT; 1mM MgCl2, 0.01% BSA) containing the desired amount of truncated GSK3polypeptide. ATP was added (specific activity 5.3 Ci/mmol) to 25 μMfinal concentration and the mixture was incubated for 20 min. at roomtemperature. The reaction was stopped by transferring 30μ onto a P81filter disc (Whatman). The disc was washed four times in 150 ml of 75 mMH₃PO₄ for 5 minute each. The filter was air dried and counted under 5 mlscintillation fluid. The specific activity was counted by determiningthe ratio of counts (in cpm) by the mass of GSK3 in the reaction (inμg).

[0074] The specific activity for construct 557 was 4.3×10⁷ cpm/μg; forconstruct 458, 2.8×10⁷ cpm/μg; and for construct 524, 2.2×10⁷ cpm/μg.TABLE 1 Mean Specific Activity Concentration Aggregation AggregationHeterogeneity Construct Purity cpm/μg N mg/ml at 4 Degrees at RT %458 >98% 2.8 × 10⁷ 35 11.5 11% @ > 2 overnight 10-20% weeksunphosphorylated 557 >98% 4.3 × 10⁷ 7 12.7 none @ > 2 overnight  5%weeks unphosphorylated 524 >98% 2.2 × 10⁷ 24 10 ND <5% unphosphorylated

Example 4 Screening for GSK3 Inhibitory Activity Using a Cell-free Assay

[0075] Compounds to be tested as GSK3 inhibitors are dissolved in DMSO,then tested for inhibition of human GSK3β. Expression of GSK3β isdescribed, for example, in Hughes et al., Eur. J. Biochem., 203:305-11(1992), which is incorporated herein by reference. An aliquot of 300 μlof substrate buffer (30 mM tris-HCl, 10 mM MgCl₂, 2 mM DTT, 3 μg/mlGSK3β) and 0.5 μM biotinylated prephosphorylated SGSG-linked CREBpeptide (Chiron Technologies PTY Ltd., Clayton, Australia) is dispensedinto wells of a 96 well polypropylene microtiter plate. 3.5 μl/well ofDMSO containing varying concentrations of each compound to be assayed orstaurosporine (a known kinase inhibitor used as a positive control, or anegative control) (i.e., DMSO only), is added and mixed thoroughly. Thereactions is then initiated by adding 50 μl/well of 1 μM unlabeled ATPand 1-2×10⁷ cpm γ³³P-labeled ATP, and the reaction is allowed to proceedfor about three hours at room temperature.

[0076] While the reaction is proceeding, streptavidin-coated Labsystems“Combiplate 8” capture plates (Labsystems, Helsinki, Finland) areblocked by incubating them with 300 μl/well of PBS containing 1% bovineserum albumin for at least one hour at room temperature. The blockingsolution is then removed by aspiration, and the capture plates arefilled with 100 μl/well of stopping reagent (50 μM ATP/20 mM EDTA).

[0077] When the three hour enzyme reaction is finished, triplicate 100μl aliquots of each reaction mix are transferred to three wellscontaining stopping solution, one well on each of the three captureplates, and the well contents are mixed well. After one hour at roomtemperature, the wells of the capture plates are emptied by aspirationand washed five times using PBS and a 12 channel Corning 430474 ELISAplate washer. Finally, 200 μl of Microscint-20 scintillation fluid isadded to each well of the plate. The plates are coated with platesealers, then left on a shaker for 30 minutes. Each capture plate iscounted in a Packard TopCount scintillation counter (Meridian,Connecticut) and the results are plotted as a function of compoundconcentration.

[0078] Compounds identified using this method can be further optimizedby testing their ability to bind to truncated GSK3 polypeptides of theinvention, using the fluorescence polarization assay, for example, fortruncated polypeptides that do not exhibit GSK3 kinase activity.Alternatively, a truncated GSK3 polypeptide of the invention can be usedin place of the native GSK3 protein.

Example 5 Screening for Inhibition of Tau Protein Phosphorylation

[0079] A. Transient Transfection of COS Cells with Expression PlasmidEncoding Truncated GSK3 and Tau Expression Plasmid Construction

[0080] COS cells are maintained in T25 tissue culture flasks in highglucose MEM medium/5% fetal bovine serum. Cells from a confluent T25flask are harvested and 80,000 cells/well are seeded into Corning 6-welltissue culture plates in a final volume of 2 ml/well of medium. Thecells are left to grow at 37° C. for 48 hours. The cells are then washedtwice in Opti-MEM containing no fetal bovine serum, and finally thecells are left in 1 ml of Opti-MEM.

[0081] Polynucleotide encoding tau protein is subcloned into plasmidpSG5 under an early SV40 promoter to generate a tau expression plasmid.The cloning of cDNA encoding tau protein is generally described inGoedert et al., EMBO Journal, 8(2):393-399 (1989), which is incorporatedherein by reference. A GSK3 expression plasmid is prepared by subcloningpolynucleotide encoding truncated GSK3 into pCG, which is an ApEVRFderivative described in Giese et al., Genes & Development, 9:995-1008(1995) and Matthias et al., Nucleic Acid Research, 17:6418 (1989), bothof which are incorporated herein by reference. The polynucleotide canencode any of the truncated GSK3 polypeptides of the invention.

[0082] The following solutions are prepared in 1.5 ml Eppendorf tubes:

[0083] Solution A: for each transfection, 2 μg of DNA (tau expressionplasmid) and 0.7 μg of DNA (GSK3 expression plasmid) are diluted into100 μl of Opti-MEM (Gibco BRL); Solution B: for each transfection, 8 μlof Lipofectamine reagent is diluted into 100 μl of Opti-MEM. The twosolutions are combined, mixed gently, and incubated at room temperaturefor 45 minutes to allow DNA-liposome complexes to form. For eachtransfection, 0.8 ml of Opti-MEM is added to the tube containing thecomplexes. The diluted solution is mixed gently and overlaid onto therinsed cells. The cells are incubated with the complexedDNA/Lipofectamine for 6 hours at 37° C. in a CO₂ incubator. Followingincubation, 1 ml of growth medium (high glucose MEM) with 20% FBS isadded to each well and incubated at 37° C. overnight. The medium isreplaced with fresh, complete medium at 18 hours following the start oftransfection, and the cells are left to grow at 37 ° C. for another 48hours.

[0084] B. Tau Phosphorylation Inhibition assay

[0085] Two hours before harvesting, 2 μl of GSK3 inhibitor dissolved inDMSO is added to each well and incubated at 37° C. After 2 hours themedium is removed and the cells are rapidly frozen on the plates on dryice and stored at −70° C. Cells are thawed on ice in the presence of 200μl lysing buffer (1% Triton® X-100, 20 mM Tris pH 7.5, 137 mM NaCl, 15%glycerol, 25 μg/ml leupeptin, 1 μg ml pepstatin-A, 1 μM PMSF, 21 μg/mlaprotinin, 50 mM NaF, 50 mM β-glycerophosphate, 15 mM sodiumpyrophosphate, 1 mM sodium orthovanadate). The contents of each well arecentrifuged at 14,000 g, 4° C. for 5 minutes and the supernatantstransferred to clean tubes. At this point the lysates may be stored at−20° C.

[0086] C. ELISA to detect phosphorylated tau in cell lysates Immulon 4strips (Dynatech) are coated with monoclonal anti-phosphorylated tau(AT8, Polymedco, Inc.) at 5 μg/ml in PBS containing Ca++ and Mg++, 100μl/well. After overnight incubation at 4° C., the strips are washedtwice with washing buffer (PBS containing 0.05% Tween® 20) and blockedwith PBS containing 1% BSA, 5% normal mouse serum and 0.05% Tween® 20 atroom temperature for 1 hour. The strips are washed 5 times with washingbuffer. Lysate (100 μl) diluted 1:10 in PBS containing 1% BSA, 0.1% NaN₃is added into each well and incubated at room temperature for 1 hour.After washing, 100 μl of 0.5 μg/ml biotinylated monoclonalanti-(non-phosphorylated) tau (HT7, Polymedco, Inc.) in PBS-BSA is addedinto each well. Strips are washed 5 times and HRP-conjugatedstreptavidin is added, incubated at room temperature for 30 minutes andwashed extensively with washing buffer. TMB substrate (Pierce) is usedfor color development and the reaction is stopped by adding an equalvolume of 0.8 M sulfuric acid. Strips are read on an ELISA plate readerusing a 450 nm filter. The concentration of compound that inhibits tauphosphorylation to 50% of the maximal level (i.e., IC₅₀) is determinedby fitting a sigmoidal curve to the plotted data.

[0087] Those skilled in the art will recognize, or be able to ascertain,using not more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such specificembodiments and equivalents are intended to be encompassed by thefollowing claims.

[0088] All patents, published patent applications, and publicationscited herein are incorporated by reference as if set forth fully herein.

1 11 1 420 PRT Homo sapiens 1 Met Ser Gly Arg Pro Arg Thr Thr Ser PheAla Glu Ser Cys Lys Pro 1 5 10 15 Val Gln Gln Pro Ser Ala Phe Gly SerMet Lys Val Ser Arg Asp Lys 20 25 30 Asp Gly Ser Lys Val Thr Thr Val ValAla Thr Pro Gly Gln Gly Pro 35 40 45 Asp Arg Pro Gln Glu Val Ser Tyr ThrAsp Thr Lys Val Ile Gly Asn 50 55 60 Gly Ser Phe Gly Val Val Tyr Gln AlaLys Leu Cys Asp Ser Gly Glu 65 70 75 80 Leu Val Ala Ile Lys Lys Val LeuGln Asp Lys Arg Phe Lys Asn Arg 85 90 95 Glu Leu Gln Ile Met Arg Lys LeuAsp His Cys Asn Ile Val Arg Leu 100 105 110 Arg Tyr Phe Phe Tyr Ser SerGly Glu Lys Lys Asp Glu Val Tyr Leu 115 120 125 Asn Leu Val Leu Asp TyrVal Pro Glu Thr Val Tyr Arg Val Ala Arg 130 135 140 His Tyr Ser Arg AlaLys Gln Thr Leu Pro Val Ile Tyr Val Lys Leu 145 150 155 160 Tyr Met TyrGln Leu Phe Arg Ser Leu Ala Tyr Ile His Ser Phe Gly 165 170 175 Ile CysHis Arg Asp Ile Lys Pro Gln Asn Leu Leu Leu Asp Pro Asp 180 185 190 ThrAla Val Leu Lys Leu Cys Asp Phe Gly Ser Ala Lys Gln Leu Val 195 200 205Arg Gly Glu Pro Asn Val Ser Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala 210 215220 Pro Glu Leu Ile Phe Gly Ala Thr Asp Tyr Thr Ser Ser Ile Asp Val 225230 235 240 Trp Ser Ala Gly Cys Val Leu Ala Glu Leu Leu Leu Gly Gln ProIle 245 250 255 Phe Pro Gly Asp Ser Gly Val Asp Gln Leu Val Glu Ile IleLys Val 260 265 270 Leu Gly Thr Pro Thr Arg Glu Gln Ile Arg Glu Met AsnPro Asn Tyr 275 280 285 Thr Glu Phe Lys Phe Pro Gln Ile Lys Ala His ProTrp Thr Lys Val 290 295 300 Phe Arg Pro Arg Thr Pro Pro Glu Ala Ile AlaLeu Cys Ser Arg Leu 305 310 315 320 Leu Glu Tyr Thr Pro Thr Ala Arg LeuThr Pro Leu Glu Ala Cys Ala 325 330 335 His Ser Phe Phe Asp Glu Leu ArgAsp Pro Asn Val Lys His Pro Asn 340 345 350 Gly Arg Asp Thr Pro Ala LeuPhe Asn Phe Thr Thr Gln Glu Leu Ser 355 360 365 Ser Asn Pro Pro Leu AlaThr Ile Leu Ile Pro Pro His Ala Arg Ile 370 375 380 Gln Ala Ala Ala SerThr Pro Thr Asn Ala Thr Ala Ala Ser Asp Ala 385 390 395 400 Asn Thr GlyAsp Arg Gly Gln Thr Asn Asn Ala Ala Ser Ala Ser Ala 405 410 415 Ser AsnSer Thr 420 2 394 PRT Homo sapiens 2 Met Glu Tyr Met Pro Met Glu Gly GlyGly Met Ser Gly Arg Pro Arg 1 5 10 15 Thr Thr Ser Phe Ala Glu Ser CysLys Pro Val Gln Gln Pro Ser Ala 20 25 30 Phe Gly Ser Met Lys Val Ser ArgAsp Lys Asp Gly Ser Lys Val Thr 35 40 45 Thr Val Val Ala Thr Pro Gly GlnGly Pro Asp Arg Pro Gln Glu Val 50 55 60 Ser Tyr Thr Asp Thr Lys Val IleGly Asn Gly Ser Phe Gly Val Val 65 70 75 80 Tyr Gln Ala Lys Leu Cys AspSer Gly Glu Leu Val Ala Ile Lys Lys 85 90 95 Val Leu Gln Asp Lys Arg PheLys Asn Arg Glu Leu Gln Ile Met Arg 100 105 110 Lys Leu Asp His Cys AsnIle Val Arg Leu Arg Tyr Phe Phe Tyr Ser 115 120 125 Ser Gly Glu Lys LysAsp Glu Val Tyr Leu Asn Leu Val Leu Asp Tyr 130 135 140 Val Pro Glu ThrVal Tyr Arg Val Ala Arg His Tyr Ser Arg Ala Lys 145 150 155 160 Gln ThrLeu Pro Val Ile Tyr Val Lys Leu Tyr Met Tyr Gln Leu Phe 165 170 175 ArgSer Leu Ala Tyr Ile His Ser Phe Gly Ile Cys His Arg Asp Ile 180 185 190Lys Pro Gln Asn Leu Leu Leu Asp Pro Asp Thr Ala Val Leu Lys Leu 195 200205 Cys Asp Phe Gly Ser Ala Lys Gln Leu Val Arg Gly Glu Pro Asn Val 210215 220 Ser Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala Pro Glu Leu Ile Phe Gly225 230 235 240 Ala Thr Asp Tyr Thr Ser Ser Ile Asp Val Trp Ser Ala GlyCys Val 245 250 255 Leu Ala Glu Leu Leu Leu Gly Gln Pro Ile Phe Pro GlyAsp Ser Gly 260 265 270 Val Asp Gln Leu Val Glu Ile Ile Lys Val Leu GlyThr Pro Thr Arg 275 280 285 Glu Gln Ile Arg Glu Met Asn Pro Asn Tyr ThrGlu Phe Lys Phe Pro 290 295 300 Gln Ile Lys Ala His Pro Trp Thr Lys ValPhe Arg Pro Arg Thr Pro 305 310 315 320 Pro Glu Ala Ile Ala Leu Cys SerArg Leu Leu Glu Tyr Thr Pro Thr 325 330 335 Ala Arg Leu Thr Pro Leu GluAla Cys Ala His Ser Phe Phe Asp Glu 340 345 350 Leu Arg Asp Pro Asn ValLys His Pro Asn Gly Arg Asp Thr Pro Ala 355 360 365 Leu Phe Asn Phe ThrThr Gln Glu Leu Ser Ser Asn Pro Pro Leu Ala 370 375 380 Thr Ile Leu IlePro Pro His Ala Arg Ile 385 390 3 361 PRT Homo sapiens 3 Met Glu Tyr MetPro Met Glu Gly Gly Gly Gly Ser Lys Val Thr Thr 1 5 10 15 Val Val AlaThr Pro Gly Gln Gly Pro Asp Arg Pro Gln Glu Val Ser 20 25 30 Tyr Thr AspThr Lys Val Ile Gly Asn Gly Ser Phe Gly Val Val Tyr 35 40 45 Gln Ala LysLeu Cys Asp Ser Gly Glu Leu Val Ala Ile Lys Lys Val 50 55 60 Leu Gln AspLys Arg Phe Lys Asn Arg Glu Leu Gln Ile Met Arg Lys 65 70 75 80 Leu AspHis Cys Asn Ile Val Arg Leu Arg Tyr Phe Phe Tyr Ser Ser 85 90 95 Gly GluLys Lys Asp Glu Val Tyr Leu Asn Leu Val Leu Asp Tyr Val 100 105 110 ProGlu Thr Val Tyr Arg Val Ala Arg His Tyr Ser Arg Ala Lys Gln 115 120 125Thr Leu Pro Val Ile Tyr Val Lys Leu Tyr Met Tyr Gln Leu Phe Arg 130 135140 Ser Leu Ala Tyr Ile His Ser Phe Gly Ile Cys His Arg Asp Ile Lys 145150 155 160 Pro Gln Asn Leu Leu Leu Asp Pro Asp Thr Ala Val Leu Lys LeuCys 165 170 175 Asp Phe Gly Ser Ala Lys Gln Leu Val Arg Gly Glu Pro AsnVal Ser 180 185 190 Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala Pro Glu Leu IlePhe Gly Ala 195 200 205 Thr Asp Tyr Thr Ser Ser Ile Asp Val Trp Ser AlaGly Cys Val Leu 210 215 220 Ala Glu Leu Leu Leu Gly Gln Pro Ile Phe ProGly Asp Ser Gly Val 225 230 235 240 Asp Gln Leu Val Glu Ile Ile Lys ValLeu Gly Thr Pro Thr Arg Glu 245 250 255 Gln Ile Arg Glu Met Asn Pro AsnTyr Thr Glu Phe Lys Phe Pro Gln 260 265 270 Ile Lys Ala His Pro Trp ThrLys Val Phe Arg Pro Arg Thr Pro Pro 275 280 285 Glu Ala Ile Ala Leu CysSer Arg Leu Leu Glu Tyr Thr Pro Thr Ala 290 295 300 Arg Leu Thr Pro LeuGlu Ala Cys Ala His Ser Phe Phe Asp Glu Leu 305 310 315 320 Arg Asp ProAsn Val Lys His Pro Asn Gly Arg Asp Thr Pro Ala Leu 325 330 335 Phe AsnPhe Thr Thr Gln Glu Leu Ser Ser Asn Pro Pro Leu Ala Thr 340 345 350 IleLeu Ile Pro Pro His Ala Arg Ile 355 360 4 483 PRT Homo sapiens 4 Met SerGly Gly Gly Pro Ser Gly Gly Gly Pro Gly Gly Ser Gly Arg 1 5 10 15 AlaArg Thr Ser Ser Phe Ala Glu Pro Gly Gly Gly Gly Gly Gly Gly 20 25 30 GlyGly Gly Pro Gly Gly Ser Ala Ser Gly Pro Gly Gly Thr Gly Gly 35 40 45 GlyLys Ala Ser Val Gly Ala Met Gly Gly Gly Val Gly Ala Ser Ser 50 55 60 SerGly Gly Gly Pro Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Pro 65 70 75 80Gly Ala Gly Thr Ser Phe Pro Pro Pro Gly Val Lys Leu Gly Arg Asp 85 90 95Ser Gly Lys Val Thr Thr Val Val Ala Thr Leu Gly Gln Gly Pro Glu 100 105110 Arg Ser Gln Glu Val Ala Tyr Thr Asp Ile Lys Val Ile Gly Asn Gly 115120 125 Ser Phe Gly Val Val Tyr Gln Ala Arg Leu Ala Glu Thr Arg Glu Leu130 135 140 Val Ala Ile Lys Lys Val Leu Gln Asp Lys Arg Phe Lys Asn ArgGlu 145 150 155 160 Leu Gln Ile Met Arg Lys Leu Asp His Cys Asn Ile ValArg Leu Arg 165 170 175 Tyr Phe Phe Tyr Ser Ser Gly Glu Lys Lys Asp GluLeu Tyr Leu Asn 180 185 190 Leu Val Leu Glu Tyr Val Pro Glu Thr Val TyrArg Val Ala Arg His 195 200 205 Phe Thr Lys Ala Lys Leu Thr Ile Pro IleLeu Tyr Val Lys Val Tyr 210 215 220 Met Tyr Gln Leu Phe Arg Ser Leu AlaTyr Ile His Ser Gln Gly Val 225 230 235 240 Cys His Arg Asp Ile Lys ProGln Asn Leu Leu Val Asp Pro Asp Thr 245 250 255 Ala Val Leu Lys Leu CysAsp Phe Gly Ser Ala Lys Gln Leu Val Arg 260 265 270 Gly Glu Pro Asn ValSer Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala Pro 275 280 285 Glu Leu Ile PheGly Ala Thr Asp Tyr Thr Ser Ser Ile Asp Val Trp 290 295 300 Ser Ala GlyCys Val Leu Ala Glu Leu Leu Leu Gly Gln Pro Ile Phe 305 310 315 320 ProGly Asp Ser Gly Val Asp Gln Leu Val Glu Ile Ile Lys Val Leu 325 330 335Gly Thr Pro Thr Arg Glu Gln Ile Arg Glu Met Asn Pro Asn Tyr Thr 340 345350 Glu Phe Lys Phe Pro Gln Ile Lys Ala His Pro Trp Thr Lys Val Phe 355360 365 Lys Ser Arg Thr Pro Pro Glu Ala Ile Ala Leu Cys Ser Ser Leu Leu370 375 380 Glu Tyr Thr Pro Ser Ser Arg Leu Ser Pro Leu Glu Ala Cys AlaHis 385 390 395 400 Ser Phe Phe Asp Glu Leu Arg Cys Leu Gly Thr Gln LeuPro Asn Asn 405 410 415 Arg Pro Leu Pro Pro Leu Phe Asn Phe Ser Ala GlyGlu Leu Ser Ile 420 425 430 Gln Pro Ser Leu Asn Ala Ile Leu Ile Pro ProHis Leu Arg Ser Pro 435 440 445 Ala Gly Thr Thr Thr Leu Thr Pro Ser SerGln Ala Leu Thr Glu Thr 450 455 460 Pro Thr Ser Ser Asp Trp Gln Ser ThrAsp Ala Thr Pro Thr Leu Thr 465 470 475 480 Asn Ser Ser 5 447 PRT Homosapiens 5 Met Ser Gly Gly Gly Pro Ser Gly Gly Gly Pro Gly Gly Ser GlyArg 1 5 10 15 Ala Arg Thr Ser Ser Phe Ala Glu Pro Gly Gly Gly Gly GlyGly Gly 20 25 30 Gly Gly Gly Pro Gly Gly Ser Ala Ser Gly Pro Gly Gly ThrGly Gly 35 40 45 Gly Lys Ala Ser Val Gly Ala Met Gly Gly Gly Val Gly AlaSer Ser 50 55 60 Ser Gly Gly Gly Pro Gly Gly Ser Gly Gly Gly Gly Ser GlyGly Pro 65 70 75 80 Gly Ala Gly Thr Ser Phe Pro Pro Pro Gly Val Lys LeuGly Arg Asp 85 90 95 Ser Gly Lys Val Thr Thr Val Val Ala Thr Leu Gly GlnGly Pro Glu 100 105 110 Arg Ser Gln Glu Val Ala Tyr Thr Asp Ile Lys ValIle Gly Asn Gly 115 120 125 Ser Phe Gly Val Val Tyr Gln Ala Arg Leu AlaGlu Thr Arg Glu Leu 130 135 140 Val Ala Ile Lys Lys Val Leu Gln Asp LysArg Phe Lys Asn Arg Glu 145 150 155 160 Leu Gln Ile Met Arg Lys Leu AspHis Cys Asn Ile Val Arg Leu Arg 165 170 175 Tyr Phe Phe Tyr Ser Ser GlyGlu Lys Lys Asp Glu Leu Tyr Leu Asn 180 185 190 Leu Val Leu Glu Tyr ValPro Glu Thr Val Tyr Arg Val Ala Arg His 195 200 205 Phe Thr Lys Ala LysLeu Thr Ile Pro Ile Leu Tyr Val Lys Val Tyr 210 215 220 Met Tyr Gln LeuPhe Arg Ser Leu Ala Tyr Ile His Ser Gln Gly Val 225 230 235 240 Cys HisArg Asp Ile Lys Pro Gln Asn Leu Leu Val Asp Pro Asp Thr 245 250 255 AlaVal Leu Lys Leu Cys Asp Phe Gly Ser Ala Lys Gln Leu Val Arg 260 265 270Gly Glu Pro Asn Val Ser Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala Pro 275 280285 Glu Leu Ile Phe Gly Ala Thr Asp Tyr Thr Ser Ser Ile Asp Val Trp 290295 300 Ser Ala Gly Cys Val Leu Ala Glu Leu Leu Leu Gly Gln Pro Ile Phe305 310 315 320 Pro Gly Asp Ser Gly Val Asp Gln Leu Val Glu Ile Ile LysVal Leu 325 330 335 Gly Thr Pro Thr Arg Glu Gln Ile Arg Glu Met Asn ProAsn Tyr Thr 340 345 350 Glu Phe Lys Phe Pro Gln Ile Lys Ala His Pro TrpThr Lys Val Phe 355 360 365 Lys Ser Arg Thr Pro Pro Glu Ala Ile Ala LeuCys Ser Ser Leu Leu 370 375 380 Glu Tyr Thr Pro Ser Ser Arg Leu Ser ProLeu Glu Ala Cys Ala His 385 390 395 400 Ser Phe Phe Asp Glu Leu Arg CysLeu Gly Thr Gln Leu Pro Asn Asn 405 410 415 Arg Pro Leu Pro Pro Leu PheAsn Phe Ser Ala Gly Glu Leu Ser Ile 420 425 430 Gln Pro Ser Leu Asn AlaIle Leu Ile Pro Pro His Leu Arg Ser 435 440 445 6 387 PRT Homo sapiens 6Ser Gly Lys Val Thr Thr Val Val Ala Thr Leu Gly Gln Gly Pro Glu 1 5 1015 Arg Ser Gln Glu Val Ala Tyr Thr Asp Ile Lys Val Ile Gly Asn Gly 20 2530 Ser Phe Gly Val Val Tyr Gln Ala Arg Leu Ala Glu Thr Arg Glu Leu 35 4045 Val Ala Ile Lys Lys Val Leu Gln Asp Lys Arg Phe Lys Asn Arg Glu 50 5560 Leu Gln Ile Met Arg Lys Leu Asp His Cys Asn Ile Val Arg Leu Arg 65 7075 80 Tyr Phe Phe Tyr Ser Ser Gly Glu Lys Lys Asp Glu Leu Tyr Leu Asn 8590 95 Leu Val Leu Glu Tyr Val Pro Glu Thr Val Tyr Arg Val Ala Arg His100 105 110 Phe Thr Lys Ala Lys Leu Thr Ile Pro Ile Leu Tyr Val Lys ValTyr 115 120 125 Met Tyr Gln Leu Phe Arg Ser Leu Ala Tyr Ile His Ser GlnGly Val 130 135 140 Cys His Arg Asp Ile Lys Pro Gln Asn Leu Leu Val AspPro Asp Thr 145 150 155 160 Ala Val Leu Lys Leu Cys Asp Phe Gly Ser AlaLys Gln Leu Val Arg 165 170 175 Gly Glu Pro Asn Val Ser Tyr Ile Cys SerArg Tyr Tyr Arg Ala Pro 180 185 190 Glu Leu Ile Phe Gly Ala Thr Asp TyrThr Ser Ser Ile Asp Val Trp 195 200 205 Ser Ala Gly Cys Val Leu Ala GluLeu Leu Leu Gly Gln Pro Ile Phe 210 215 220 Pro Gly Asp Ser Gly Val AspGln Leu Val Glu Ile Ile Lys Val Leu 225 230 235 240 Gly Thr Pro Thr ArgGlu Gln Ile Arg Glu Met Asn Pro Asn Tyr Thr 245 250 255 Glu Phe Lys PhePro Gln Ile Lys Ala His Pro Trp Thr Lys Val Phe 260 265 270 Lys Ser ArgThr Pro Pro Glu Ala Ile Ala Leu Cys Ser Ser Leu Leu 275 280 285 Glu TyrThr Pro Ser Ser Arg Leu Ser Pro Leu Glu Ala Cys Ala His 290 295 300 SerPhe Phe Asp Glu Leu Arg Cys Leu Gly Thr Gln Leu Pro Asn Asn 305 310 315320 Arg Pro Leu Pro Pro Leu Phe Asn Phe Ser Ala Gly Glu Leu Ser Ile 325330 335 Gln Pro Ser Leu Asn Ala Ile Leu Ile Pro Pro His Leu Arg Ser Pro340 345 350 Ala Gly Thr Thr Thr Leu Thr Pro Ser Ser Gln Ala Leu Thr GluThr 355 360 365 Pro Thr Ser Ser Asp Trp Gln Ser Thr Asp Ala Thr Pro ThrLeu Thr 370 375 380 Asn Ser Ser 385 7 351 PRT Homo sapiens 7 Ser Gly LysVal Thr Thr Val Val Ala Thr Leu Gly Gln Gly Pro Glu 1 5 10 15 Arg SerGln Glu Val Ala Tyr Thr Asp Ile Lys Val Ile Gly Asn Gly 20 25 30 Ser PheGly Val Val Tyr Gln Ala Arg Leu Ala Glu Thr Arg Glu Leu 35 40 45 Val AlaIle Lys Lys Val Leu Gln Asp Lys Arg Phe Lys Asn Arg Glu 50 55 60 Leu GlnIle Met Arg Lys Leu Asp His Cys Asn Ile Val Arg Leu Arg 65 70 75 80 TyrPhe Phe Tyr Ser Ser Gly Glu Lys Lys Asp Glu Leu Tyr Leu Asn 85 90 95 LeuVal Leu Glu Tyr Val Pro Glu Thr Val Tyr Arg Val Ala Arg His 100 105 110Phe Thr Lys Ala Lys Leu Thr Ile Pro Ile Leu Tyr Val Lys Val Tyr 115 120125 Met Tyr Gln Leu Phe Arg Ser Leu Ala Tyr Ile His Ser Gln Gly Val 130135 140 Cys His Arg Asp Ile Lys Pro Gln Asn Leu Leu Val Asp Pro Asp Thr145 150 155 160 Ala Val Leu Lys Leu Cys Asp Phe Gly Ser Ala Lys Gln LeuVal Arg 165 170 175 Gly Glu Pro Asn Val Ser Tyr Ile Cys Ser Arg Tyr TyrArg Ala Pro 180 185 190 Glu Leu Ile Phe Gly Ala Thr Asp Tyr Thr Ser SerIle Asp Val Trp 195 200 205 Ser Ala Gly Cys Val Leu Ala Glu Leu Leu LeuGly Gln Pro Ile Phe 210 215 220 Pro Gly Asp Ser Gly Val Asp Gln Leu ValGlu Ile Ile Lys Val Leu 225 230 235 240 Gly Thr Pro Thr Arg Glu Gln IleArg Glu Met Asn Pro Asn Tyr Thr 245 250 255 Glu Phe Lys Phe Pro Gln IleLys Ala His Pro Trp Thr Lys Val Phe 260 265 270 Lys Ser Arg Thr Pro ProGlu Ala Ile Ala Leu Cys Ser Ser Leu Leu 275 280 285 Glu Tyr Thr Pro SerSer Arg Leu Ser Pro Leu Glu Ala Cys Ala His 290 295 300 Ser Phe Phe AspGlu Leu Arg Cys Leu Gly Thr Gln Leu Pro Asn Asn 305 310 315 320 Arg ProLeu Pro Pro Leu Phe Asn Phe Ser Ala Gly Glu Leu Ser Ile 325 330 335 GlnPro Ser Leu Asn Ala Ile Leu Ile Pro Pro His Leu Arg Ser 340 345 350 8 15PRT Artificial Sequence N-terminus addition sequence 8 Glu Phe Met ProThr Glu Ala Met Ala Ala Pro Lys Arg Val Ile 1 5 10 15 9 9 PRT ArtificialSequence N-terminus addition sequence 9 Glu Tyr Met Pro Met Glu Gly GlyGly 1 5 10 6 PRT Artificial Sequence elution peptide 10 Glu Tyr Met ProThr Asp 1 5 11 5 PRT Artificial Sequence Peptide substratephosphorylatable by GSK3 11 Ser Xaa Xaa Xaa Ser 1 5

1. An isolated nucleic acid comprising a polynucleotide encoding apolypeptide consisting essentially of SEQ ID NO:2, wherein saidpolypeptide will crystallize and will have at least one biologicalactivity selected from the group consisting of (a) binding a GSK3inhibitor; and (b) kinase activity.
 2. An isolated nucleic acidcomprising a polynucleotide encoding a polypeptide consistingessentially of SEQ ID NO:3, wherein said polypeptide will crystallizeand will have at least one biological activity selected from the groupconsisting of (a) binding a GSK3 inhibitor; and (b) kinase activity. 3.A vector comprising the polynucleotide of claim I or claim
 2. 4. Apolypeptide comprising between about 250 and 419 contiguous amino acidsof SEQ ID NO:1, wherein said polypeptide is phosphorylated on tyrosine216, said polypeptide will crystallize, and said polypeptide will haveat least one biological activity selected from the group consisting of(a) binding a GSK3 inhibitor; and (b) kinase activity.
 5. A polypeptideconsisting essentially of between about 278 and 419 contiguous aminoacids of SEQ ID NO:1, wherein said polypeptide exhibits at least 1% ofthe kinase activity of human GSK3β.
 6. A polypeptide consistingessentially of between about 285 and 384 contiguous amino acids of SEQID NO:1, wherein said polypeptide exhibits at least 1% of the kinaseactivity of human GSK3β.
 7. A polypeptide consisting essentially ofbetween about 351 and 384 contiguous amino acids of SEQ ID NO:1, whereinsaid polypeptide exhibits at least 1% of the kinase activity of humanGSK3β.
 8. A polypeptide consisting of the amino acid sequence of SEQ IDNO:2.
 9. A polypeptide consisting of the amino acid sequence of SEQ IDNO:3.
 10. An isolated nucleic acid comprising a polynucleotide encodinga polypeptide consisting essentially of SEQ ID NO:5, wherein saidpolypeptide will crystallize and will have at least one biologicalactivity selected from the group consisting of (a) binding to a GSK3inhibitor; and (b) kinase activity.
 11. The nucleic acid of claim 10wherein said polypeptide is phosphorylated on tyrosine
 279. 12. A vectorcomprising the polynucleotide of claim 10 or claim
 11. 13. A polypeptidecomprising between about 182 and 482 contiguous amino acids of SEQ IDNO:4, wherein said polypeptide will crystallize, and said polypeptidewill have at least one biological activity selected from the groupconsisting of (a) binding a GSK3 inhibitor; and (b) kinase activity. 14.A polypeptide consisting essentially of between about 182 and 386contiguous amino acids of SEQ ID NO:4, wherein said polypeptide exhibitsat least 1% of the kinase activity of human GSK3α.
 15. A polypeptideconsisting essentially of between about 182 and 351 contiguous aminoacids of SEQ ID NO:4, wherein said polypeptide exhibits at least 1% ofthe kinase activity of human GSK3α.
 16. A polypeptide consistingessentially of contiguous amino acids S⁹⁷ to S⁴⁴⁷ of SEQ ID NO:1.
 17. Apolypeptide consisting essentially of the amino acid sequence of SEQ IDNO:5.
 18. A polynucleotide encoding a polypeptide consisting essentiallyof SEQ ID NO:6.
 19. A polypeptide consisting essentially of the aminoacid sequence of SEQ ID NO:6.
 20. A polynucleotide encoding apolypeptide consisting essentially of SEQ ID NO:7.
 21. A polypeptideconsisting essentially of the amino acid sequence of SEQ ID NO:7.
 22. Apolynucleotide encoding a non-phosphorylated human GSK3 polypeptide,wherein said non-phosphorylated polypeptide differs from native GSK3 inat least one and not more than ten amino acids.
 23. The polynucleotideof claim 22 wherein tyrosine at position 216 of SEQ ID NO:1 issubstituted for by a non-phosphorylatable amino acid.
 24. Thepolynucleotide of claim 23 wherein said non-phosphorylatable amino acidis phenylalanine.
 25. The polynucleotide of claim 22 wherein tyrosine atposition 279 of SEQ ID NO:4 is substituted for by a non-phosphorylatableamino acid.
 26. The polynucleotide of claim 25 wherein saidnon-phosphorylatable amino acid is phenylalanine.